Membrane fusion is a fundamental process involved in diverse cellular events such as fertilization and neurosecretion. Biological membrane fusion relies on proteins to drive membrane merger and is likely facilitated by specific lipid geometries in vivo. A family of integral membrane proteins collectively known as SNAREs mediates the fusion of intracellular transport vesicles. While SNAREs pair in specific ways to provide the mechanical energy to drive fusion, the delicate interplay of regulatory elements that orchestrate this event in space and time remain elusive. We use a combination of in vitro fusion assays with purified yeast SNARE proteins and lipids, yeast genetics, and cell biology to examine mechanistic details of membrane fusion during exocytosis. SNAREs and regulatory proteins will be manipulated in vivo and their specific effects on membrane fusion can be directly analyzed in vitro. A molecular appreciation of the dynamic protein-protein interactions that execute and regulate membrane fusion during secretion could potentially lead to important therapeutic targets. We begin by examining the regulatory role of Sec1p in yeast exocytosis. Sec1p binds primarily to the yeast t-SNARE complex and directly stimulates membrane fusion. We will investigate how Sec1p binds to the t-SNARE complex and fully assembled ternary SNARE complex and the functional consequences of this binding. We will also determine the mechanistic basis for Sec1p stimulation by comparing neuronal Sec1 (n-Sec1) with its yeast counterpart Sec1p. Next, we will dissect plasma membrane t-SNARE complex function in vitro and in vivo. The Sso1p N-terminal regulatory domain (NRD) is dispensable in vitro but required in vivo. We will determine the function of the Sso1p N-terminal regulatory domain testing the hypothesis that the Sso1p NRD serves a chaperone function for the Sso1p core H3 domain. Additionally, we examine the function of the Sso1p polybasic juxtamembrane region, a conserved sequence in all plasma membrane SNAREs. Third, we analyze membrane fusion driven by the sporulation specific t-SNARE light chain Spo20p, which requires the addition of phosphatidic acid to the bilayer for efficient fusion. We explore the mechanistic basis for the difference between the t-SNARE light chains Sec9p and Spo20p by examining lipid requirements and structural stability of each t-SNARE complex. Finally, we compare SNARE-mediated fusion with purified organelles and synthetic liposomes. Purified secretory vesicles and inverted plasma membrane vesicles will be used with existing synthetic proteoliposomes to study fusion with native membranes in an effort to reveal differences in fusion with SNARE mutants in vitro and in vivo.